Image flow cytometry color separation adjusting system and correction method thereof

Abstract

The invention relates to the technical field of flow cytometry, and particularly discloses an image flow cytometry color separation adjusting system and a correction method thereof, and the system comprises a light source assembly which is used for generating parallel light; the beam splitter prism is used for splitting the parallel light into reflected light and transmitted light; the color camera is used for collecting the reflected light to generate a reflected light image; the dichroscope stack is provided with a plurality of dichroscope lenses with adjustable orientation angles and is used for outputting a plurality of emergent light beams with different wavelengths according to the transmission light; the high-speed camera is used for collecting the emergent light beam to generate a plurality of sections of narrow-band emergent light images; the processing module is used for processing the multi-section narrow-band emergent light image into a microcosmic multi-color image so as to carry out difference comparison on the microcosmic multi-color image and the reflected light image to realize color separation correction adjustment; according to the system, color separation correction adjustment can be conveniently and visually completed, and the adjustment precision of color separation correction of the streaming area array image acquisition system is effectively improved.

Description

Technical Field

[0001] The present application relates to the technical field of flow cytometers, and in particular to a color separation adjustment system for an image flow cytometer and a correction method thereof. Background Art

[0002] Image flow cytometry is an emerging high-speed particle image acquisition method. Compared with traditional flow cytometry, it not only maintains the advantages of high-throughput and high-speed detection, but also realizes the detection of high-content information of cells and avoids the need for fluorescence compensation. Therefore, it has become a new hot spot in the development of flow cytometers.

[0003] Currently, image flow cytometry is mainly divided into two types: point scanning and area array detection. Among them, the area array detection method uses the area array sensor of a high-speed camera to directly collect image fluorescence. It has a simple optical path and is easy to debug, but it has high requirements for the fluorescence color separation imaging structure.

[0004] In the streaming array image acquisition system, in order to reduce costs and simplify the optical path, a monochrome camera is usually used to acquire multi-color fluorescence images, and the merging of multi-channel particle images is achieved through back-end algorithms and image processing. However, this process requires extremely high accuracy of the fluorescence color separation imaging structure, otherwise it will affect the efficiency of image transmission, cropping, and merging, and even lead to the inability to obtain a correct reconstructed image.

[0005] At present, the commonly used color separation imaging structure schemes mostly use dichroic mirror stacks, but there are many problems and inconveniences in the adjustment and control process, especially the cumbersome correction process, the lack of unified correction standards, and the difficulty in intuitively verifying the correctness of the reconstructed image.

[0006] There is currently no effective technical solution to the above problems. Summary of the invention

[0007] The purpose of the present application is to provide an image flow cytometer color separation adjustment system and a correction method thereof, so as to complete the color separation correction adjustment conveniently and intuitively, and improve the adjustment accuracy of the color separation correction of the flow array image acquisition system.

[0008] In a first aspect, the present application provides a color separation adjustment system for an image flow cytometer, the system comprising: A light source assembly, used for generating parallel light; A beam splitter prism, used to split the parallel light into reflected light and transmitted light; A color camera, used for collecting the reflected light to generate a reflected light image; A dichroic mirror stack, comprising a plurality of dichroic lenses with adjustable orientation angles, for outputting a plurality of outgoing light beams of different wavelengths according to the transmitted light; A high-speed camera, used for collecting the outgoing light beam to generate multiple narrow-band outgoing light images; The processing module is used to process the multiple narrow-band emission light images into microscopic multi-color images so as to compare the differences with the reflected light images to realize color separation correction and adjustment.

[0009] The image flow cytometer color separation adjustment system of the present application is provided with a dichroic prism and a color camera to obtain a reflected light image that can represent the imaging effect of an actual color image, and this can be used as a unified correction standard, so that the image flow cytometer color separation adjustment system of the present application can evaluate the imaging quality of the microscopic multi-color image by comparing the difference between the microscopic multi-color image and the reflected light image, and use this as a reference to adjust the orientation angle of each dichroic lens in the dichroic mirror stack, so as to complete the color separation correction adjustment conveniently and intuitively, and can effectively improve the adjustment accuracy of the color separation correction of the flow array image acquisition system.

[0010] The image flow cytometer color separation adjustment system, wherein the dichroic mirror stack comprises a base frame, an adjustment frame, a first adjustment component and a plurality of adjustable lens components; The adjustable lens assemblies are all mounted on the adjustment frame, the adjustment frame is mounted on the base frame, and the first adjustment assembly is used to adjust the position of the adjustment frame on the base frame to synchronously change the orientation angle of the adjustable lens assembly relative to the dichroic prism; Each of the adjustable lens assemblies includes a dichroic lens with an adjustable orientation angle.

[0011] The image flow cytometer color separation adjustment system of the present application can adjust the orientation of the entire adjustment frame through the first adjustment component, thereby realizing synchronous adjustment of the orientation angles of all color separation light beams, and then changing the imaging position of the multi-segment narrow-band output light image formed by all the color separation light beams, and the orientation angle of the dichroic mirror in each adjustable lens component can be adjusted independently, so that the position of the color separation light spot corresponding to each narrow band in the multi-segment narrow-band output light image can be independently adjusted, thereby achieving the purpose of color separation adjustment.

[0012] The image flow cytometer color separation adjustment system, wherein the adjustable lens assembly includes a lens clip, the dichroic lens and a second adjustment assembly, the dichroic lens is fixed on the lens clip, the lens clip is installed on the adjustment frame, and the second adjustment assembly is used to adjust the position of the lens clip on the adjustment frame to change the orientation angle of the corresponding dichroic lens relative to the beam splitting prism.

[0013] The image flow cytometer color separation adjustment system of the present application can adjust the orientation of the lens clamp through the second adjustment component, thereby independently adjusting the position of the color separation light spot corresponding to each narrow band in the multi-segment narrow band output light image, thereby achieving the purpose of color separation adjustment.

[0014] The image flow cytometer color separation adjustment system, wherein the process of processing the multiple narrow-band emission light images into microscopic multi-color images includes: The multiple narrow-band emission light images are sequentially subjected to color separation, cropping, color restoration and merging processing to obtain the microscopic multi-color image.

[0015] The image flow cytometer color separation and adjustment system, wherein the light source component comprises an optical fiber point light source, a light source collimation component and a light source filter which are arranged in sequence.

[0016] The image flow cytometer color separation adjustment system, wherein the light source collimation assembly includes a collimating lens, an optical fiber fixing sleeve, a collimating lens frame, a fixing clamp ring and a third adjustment assembly; The optical fiber point light source is fixed on the optical fiber fixing sleeve, the optical fiber fixing sleeve is fixedly connected to the collimating lens frame, the collimating lens is fixed on the collimating lens frame through a fixing clamp, and the collimating lens frame is height-adjustably mounted on the third adjustment component.

[0017] In the image flow cytometer color separation adjustment system, a focusing lens assembly is provided between the beam splitting prism and the color camera and between the dichroic mirror stack and the high-speed camera.

[0018] In a second aspect, the present application further provides a correction method for a color separation adjustment system of an image flow cytometer, which is applied in a color separation adjustment system of an image flow cytometer, and the system comprises: A light source assembly, used for generating parallel light; A beam splitter prism, used to split the parallel light into reflected light and transmitted light; A color camera, used for collecting the reflected light to generate a reflected light image; A dichroic mirror stack, comprising a plurality of dichroic lenses with adjustable orientation angles, for outputting a plurality of outgoing light beams of different wavelengths according to the transmitted light; A high-speed camera, used for collecting the outgoing light beam to generate multiple narrow-band outgoing light images; A processing module, used for processing the multiple narrow-band emission light images into microscopic multi-color images, so as to compare the difference with the reflected light image to realize color separation correction and adjustment; The method comprises the steps of: S1. sequentially adjusting the orientation angles of the dichroic mirrors in the dichroic mirror stack based on the preset positions and accuracy requirements, so as to correct and adjust the positions of the narrow-band graphics in the multi-segment narrow-band emergent light images; S2, comparing the difference between the microscopic multi-color image after correction and adjustment and the corresponding reflected light image, if the difference is as expected, then end the correction, otherwise return to step S1.

[0019] The correction method of the color separation adjustment system of the image flow cytometer of the present application realizes the color separation correction adjustment of the color separation adjustment system of the image flow cytometer by means of secondary verification. The method realizes the secondary verification by comparing the image difference between the reflected light image which is based on the imaging effect representing the actual color image and used as the unified correction standard and the microscopic multi-color color image which is reconstructed based on the multi-segment narrow-band output light image after the correction adjustment, so as to intuitively evaluate the imaging quality of the microscopic multi-color color image. When it does not meet the expectations, the orientation angle of each dichroic lens in the dichroic mirror stack is adjusted based on this, so as to complete the color separation correction adjustment conveniently and intuitively, and can effectively improve the adjustment accuracy of the color separation correction of the flow array image acquisition system.

[0020] In the calibration method of the color separation adjustment system of the image flow cytometer, during the execution of step S1, a micro-target is provided between the light source assembly and the beam splitter prism.

[0021] The light spot formed by the output of the light source component may be of poor quality, which may easily lead to blurred or missing edges of the light spot, making it impossible to use it as a reference image. In these cases, the correction method of the image flow cytometer color separation adjustment system of the present application introduces a micro-target as a reference image to ensure that the color separation correction adjustment process can proceed smoothly.

[0022] In the calibration method of the color separation adjustment system of the image flow cytometer, when step S2 is executed for a preset number of times, if the difference still does not meet expectations, the dichroic mirror stack is replaced or the preset position is modified, and then the process returns to step S1.

[0023] As can be seen from the above, the present application provides an image flow cytometer color separation adjustment system and a correction method thereof, wherein the image flow cytometer color separation adjustment system obtains a reflected light image that can represent the imaging effect of an actual color image by setting a dichroic prism and a color camera, and can use this as a unified correction standard, so that the image flow cytometer color separation adjustment system of the present application can evaluate the imaging quality of the microscopic multi-color image by comparing the difference between the microscopic multi-color image and the reflected light image, and use this as a reference to adjust the orientation angle of each dichroic lens in the dichroic mirror stack, so as to complete the color separation correction adjustment conveniently and intuitively, which can effectively improve the adjustment accuracy of the color separation correction of the flow array image acquisition system. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A simplified structural diagram of a color separation and adjustment system for an image flow cytometer provided in an embodiment of the present application.

[0025] Figure 2 This is a simplified structural schematic diagram of the color separation and adjustment system of the image flow cytometer provided in an embodiment of the present application after adding a micro-target.

[0026] Figure 3 A detailed structural diagram of the color separation and adjustment system of the image flow cytometer provided in an embodiment of the present application.

[0027] Figure 4 Schematic diagram of the exploded structure of the fiber optic point light source and light source collimation component.

[0028] Figure 5 Schematic diagram of the exploded structure of the dichroic mirror stack.

[0029] Figure 6 This is a flow chart of a correction method for a color separation adjustment system of an image flow cytometer provided in an embodiment of the present application.

[0030] Figure numerals: 1. light source assembly; 2. beam splitter; 3. color camera; 4. dichroic mirror stack; 5. high-speed camera; 6. processing module; 7. focusing lens assembly; 8. fifth adjustment assembly; 9. micro target; 11. optical fiber point light source; 12. light source collimation assembly; 13. light source filter; 121. collimating lens; 122. optical fiber fixing sleeve; 123. collimating lens frame; 124. fixing clamp; 125. third adjustment assembly; 126. fourth adjustment assembly; 1251. base frame; 1252. mounting shaft; 1253. tightening bolt; 41. base frame; 42. adjustment frame; 43. first adjustment assembly; 44. adjustable lens assembly; 441. lens clamp; 442. dichroic lens; 443. second adjustment assembly. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0032] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.

[0033] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or mutual communication; it can be a direct connection, or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0034] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include that the first and second features are in direct contact, or may include that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, a first feature being "above", "above" and "above" a second feature includes that the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. A first feature being "below", "below" and "below" a second feature includes that the first feature is directly below and obliquely below the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0035] The disclosure below provides many different embodiments or examples to realize different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplicity and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed. In addition, the present invention provides various specific examples of processes and materials, but those of ordinary skill in the art can be aware of the application of other processes and / or the use of other materials.

[0036] First, please refer to Figure 1-Figure 5 Some embodiments of the present application provide a color separation adjustment system for an image flow cytometer, the system comprising: A light source assembly 1, used to generate parallel light; A beam splitter prism 2, used for splitting parallel light into reflected light and transmitted light; A color camera 3, used for collecting reflected light to generate a reflected light image; A dichroic mirror stack 4, having a plurality of dichroic lenses 442 with adjustable orientation angles, for outputting a plurality of outgoing light beams of different wavelengths according to the transmitted light; A high-speed camera 5 is used to collect the outgoing light beam to generate multiple narrow-band outgoing light images; The processing module 6 is used to process the multiple narrow-band emission light images into microscopic multi-color images, so as to compare the difference with the reflected light image to realize color separation correction and adjustment.

[0037] Specifically, the image flow cytometer color separation adjustment system is applied in the flow array image acquisition system, which aims to provide the flow array image acquisition system with a more intuitive and convenient color separation correction adjustment means with visual correction standards.

[0038] More specifically, in the above structure, the light source assembly 1, the dichroic mirror stack 4, the high-speed camera 5 and the processing module 6 constitute an acquisition module in the flow array image acquisition system. By placing a sample delivery module (such as a fluid pool) with an object to be acquired in the light source assembly 1 and the dichroic mirror stack 4, the flow data corresponding to the object to be acquired can be obtained through the processing module 6. On this basis, the image flow cytometer color separation adjustment system of the embodiment of the present application adds a beam splitter prism 2 and a color camera 3, wherein the beam splitter prism 2 can divide the parallel light into reflected light and transmitted light based on a preset beam splitting ratio without changing the polarization state, wherein the transmitted light is projected into the high-speed camera 5 through the dichroic mirror stack 4 to generate a multi-segment narrow-band output light image, and the reflected light is directly projected The light is projected into the color camera 3 for imaging to generate a colored reflected light image; the multi-segment narrow-band emergent light images collected by the high-speed camera 5 are black and white images; the dichroic mirror stack 4 uses a plurality of dichroic lenses 442 with different orientation angles thereon to reflect the transmitted light, so as to divide the emergent light into a plurality of narrow-band light beams with different emergent angles according to different narrow bands; these narrow-band light beams hit different positions of the sensor of the high-speed camera 5 to form a plurality of color-separated light spots; the high-speed camera 5 collects these color-separated light spots through its sensor to obtain a multi-segment narrow-band emergent light image; the light spot image objects at different positions on the multi-segment narrow-band emergent light image correspond to different colors; therefore, the processing module 6 can restore the multi-segment narrow-band emergent light image based on a preset algorithm to obtain a microscopic multi-color image. The quality of the restoration effect is mainly affected by the accuracy of the imaging position of the color separation spot. The image flow cytometer color separation adjustment system of the embodiment of the present application synchronously generates a microscopic multi-color color image and a reflected light image, so that the microscopic multi-color color image and the reflected light image have a paired relationship. The reflected light image represents the imaging effect of the actual color image. Therefore, by comparing the difference between the microscopic multi-color color image and the reflected light image, the imaging quality of the microscopic multi-color color image can be evaluated. The more similar the two are, the more accurate the imaging position of the corresponding color separation spot in the current microscopic multi-color color image is, that is, the higher the imaging quality is. The user can intuitively adjust the orientation angle of each dichroic lens 442 in the dichroic mirror stack 4 according to the difference between the two types of images to change the restoration effect of the microscopic multi-color color image, thereby conveniently completing the color separation correction adjustment.

[0039] It should be noted that the process of comparing the difference between the microscopic multi-color image and the reflected light image can be done by manually judging the difference between the two, or by using the processing module 6 to determine the difference between the two based on a preset similarity algorithm or difference algorithm and outputting the corresponding image similarity of the two types of images in real time.

[0040] It should be noted that after completing the color separation correction, the user can selectively remove the beam splitter prism 2 to improve the imaging brightness of the microscopic multi-color image.

[0041] The color separation adjustment system of the image flow cytometer of the embodiment of the present application sets a dichroic prism 2 and a color camera 3 to obtain a reflected light image that can represent the imaging effect of the actual color image, and can use this as a unified correction standard, so that the color separation adjustment system of the image flow cytometer of the embodiment of the present application can evaluate the imaging quality of the microscopic multi-color image by comparing the difference between the microscopic multi-color image and the reflected light image, and use this as a reference to adjust the orientation angle of each dichroic lens 442 in the dichroic mirror stack 4, so as to complete the color separation correction adjustment conveniently and intuitively, which can effectively improve the adjustment accuracy of the color separation correction of the flow array image acquisition system.

[0042] In some preferred embodiments, the dichroic mirror stack 4 includes a base frame 41, an adjustment frame 42, a first adjustment assembly 43, and a plurality of adjustable lens assemblies 44; The adjustable lens assemblies 44 are all mounted on the adjustment frame 42, the adjustment frame 42 is mounted on the base frame 41, and the first adjustment assembly 43 is used to adjust the position of the adjustment frame 42 on the base frame 41 to synchronously change the orientation angle of the adjustable lens assembly 44 relative to the beam splitter prism 2; Each adjustable lens assembly 44 includes a dichroic lens 442 whose orientation angle is adjustable.

[0043] Specifically, in this embodiment, there is a non-zero angle between the mirror surfaces of adjacent dichroic lenses 442, which enables different dichroic lenses 442 to reflect light of different narrow bands at different angles to perform color separation reflection on the transmitted light to form color separation light beams. These color separation light beams form position-separated color separation light spots on the sensor target of the high-speed camera 5, thereby obtaining multiple narrow-band output light images.

[0044] More specifically, the color separation adjustment system of the image flow cytometer of the embodiment of the present application can adjust the orientation of the entire adjustment frame 42 through the first adjustment component 43, thereby realizing the synchronous adjustment of the orientation angles of all color separation light beams, and then changing the imaging position of the multi-segment narrow-band output light image formed by all the color separation light beams, and the orientation angle of the dichroic mirror in each adjustable lens component 44 can be adjusted independently, so that the position of the color separation light spot corresponding to each narrow band in the multi-segment narrow-band output light image can be independently adjusted, thereby achieving the purpose of color separation adjustment.

[0045] It should be noted that, in the embodiment of the present application, the orientation angle of each dichroic lens 442 can be adjusted in the pitch angle and the yaw angle direction, so that the corresponding narrow-band color separation light spots in the multi-segment narrow-band output light image can be freely adjusted in position on the target that appears to be a plane.

[0046] In some preferred embodiments, the adjustable lens assembly 44 includes a lens clip 441, a dichroic lens 442 and a second adjustment assembly 443. The dichroic lens 442 is fixed on the lens clip 441, and the lens clip 441 is installed on the adjustment frame 42. The second adjustment assembly 443 is used to adjust the position of the lens clip 441 on the adjustment frame 42 to change the orientation angle of the corresponding dichroic lens 442 relative to the beam splitter prism 2.

[0047] Specifically, the dichroic lens 442 can be fixed on the corresponding lens clip 441 by optical adhesive.

[0048] More specifically, the image flow cytometer color separation adjustment system of the embodiment of the present application can adjust the orientation of the lens clip 441 through the second adjustment component 443, thereby independently adjusting the position of the color separation spot corresponding to each narrow band in the multi-segment narrow band output light image, thereby achieving the purpose of color separation adjustment.

[0049] More specifically, in the embodiment of the present application, there are preferably three adjustable lens assemblies 44, and the corresponding three dichroic lenses 442 can separate parallel light into three narrow-band color-separated light beams, and are particularly suitable for separating the light beam into three color-separated light beams of red, green, and blue (matching the RGB relationship), so that the processing module 6 can reconstruct a microscopic multi-color image that matches the color of the reflected light image captured by the color camera 3 based on the multiple narrow-band output light images, thereby ensuring the effectiveness of the reflected light image as a unified correction standard, thereby improving the accuracy of the difference contrast, and further improving the accuracy of the color separation correction adjustment.

[0050] In some preferred embodiments, the first adjustment component 43 includes an adjustment screw and a first spring. The base frame 41 is connected to the adjustment frame 42 through the first spring. The adjustment screw is threadedly connected to the base frame 41, and one end thereof is pressed against the adjustment frame 42 under the tension of the first spring.

[0051] Specifically, in this embodiment, the user can change the protruding length of the adjusting screw on the base frame 41 by turning the adjusting screw, so that the adjusting frame 42 tightened by the adjusting screw can be deflected relative to the base frame 41 with the support of the first spring, thereby realizing the adjustment of the orientation of the entire adjusting frame 42. The structural composition of the above-mentioned first adjusting component 43 has the advantages of high adjustment accuracy and convenient adjustment.

[0052] In some preferred embodiments, the second adjustment component 443 includes a second spring, a ball and an adjustment knob. The adjustment frame 42 is connected to the lens clamp 441 through the second spring. Adjacent lens clamps 441 are spaced apart by ball bearings. The adjustment knob is threadedly connected to the lens clamp 441, and one end thereof is pressed against the adjacent lens clamp 441 under the tension of the second spring.

[0053] Specifically, the working principle of the second adjustment component 443 is similar to that of the first adjustment component 43. The user can change the protruding length of the adjustment knob on the lens clamp 441 by turning the adjustment knob, so that the adjacent lens clamp 441 pressed by the adjustment knob can be deflected relative to the lens clamp 441 with the support of the ball, thereby realizing the separate adjustment of the orientation of the lens clamp 441 and achieving the purpose of color separation adjustment. The structural composition of the above-mentioned second adjustment component 443 has the advantages of high adjustment accuracy and convenient adjustment.

[0054] It should be noted that if Figure 5 As shown, in the embodiment in which there are three adjustable lens assemblies 44, adjustment knobs are only provided on the outermost and innermost lens clip bodies 441.

[0055] In some preferred embodiments, the process of processing multiple narrow-band emission light images into microscopic multi-color images includes: The multiple narrow-band output light images are sequentially subjected to color separation, cropping, color restoration and merging processes to obtain a microscopic multi-color image.

[0056] Specifically, under the color separation effect of the dichroic mirror stack 4, the transmitted light is divided into a plurality of narrow-band light beams with different emission angles according to different narrow bands, and is focused onto different positions of the sensor target surface of the high-speed camera 5, thereby realizing color separation imaging (i.e., collecting multi-channel multi-color light beams by collecting one image at a time to excite and obtain multi-color fluorescence). Since the multi-segment narrow-band output light image is a black and white image, the processing module 6 needs to use color separation, cropping, color restoration and merging processing methods to restore the multi-segment narrow-band output light image to a microscopic multi-color color image that can be compared with the reflected light image; wherein the cropping process is to collect a plurality of small images representing different narrow-band light beams from the entire multi-segment narrow-band output light image collected by the entire high-speed camera 5 based on a preset image division rule, and at the same time remove useless pixel data to improve the processing efficiency of subsequent processing steps; the restoration process is to restore each small image to its corresponding color according to the set color separation band (determined based on the specifications of the dichroic mirror stack 4), such as restoring the small image corresponding to the green light band into a green image; the merging process is to merge the various color images according to the pixel position of the small image after each color restoration (that is, the geometric center pixel position of each color image coincides) to obtain the desired multi-color image, that is, the microscopic multi-color color image.

[0057] In some preferred embodiments, the light source assembly 1 includes a fiber optic point light source 11 , a light source collimating assembly 12 , and a light source filter 13 , which are arranged in sequence.

[0058] Specifically, in this embodiment, the fiber optic point light source 11 is a point light source that outputs white light through optical fiber transmission. In the embodiment of the present application, it preferably includes a quartz halogen lamp and an optical fiber that generate white light. The white light output by the quartz halogen lamp is transmitted to the end output port through the optical fiber, and then diverges and outputs as a point light source.

[0059] In other embodiments, the optical fiber point light source 11 may also be replaced by an LED white light point light source, and a point light source with a smaller diameter and a better light spot shape needs to be selected accordingly.

[0060] It should be noted that the selection of the optical fiber point light source 11 should match the selection of the light source filter 13 and the dichroic mirror stack 4, so as to ensure that a color separation light spot that meets the acquisition requirements of the streaming area array image acquisition system can be finally generated.

[0061] More specifically, the fiber optic point light source 11 outputs divergent white light, which is then processed into wide-band parallel light by the light source collimation component 12 for subsequent system illumination or directly as an obtainable image profile. The light source filter 13 can then filter out stray light in the parallel light and limit the wavelength range of the parallel light to modify it into parallel light that meets the acquisition requirements of the streaming array image acquisition system.

[0062] In some preferred embodiments, the light source collimating assembly 12 includes a collimating lens 121, an optical fiber fixing sleeve 122, a collimating lens holder 123, a fixing clamp 124 and a third adjusting assembly 125; The optical fiber point light source 11 is fixed on the optical fiber fixing sleeve 122, the optical fiber fixing sleeve 122 is fixedly connected to the collimating lens frame 123, the collimating lens 121 is fixed on the collimating lens frame 123 through a fixing clamp 124, and the collimating lens frame 123 is height-adjustably mounted on the third adjustment component 125.

[0063] Specifically, the above-mentioned structural combination can make the installation position of the optical fiber point light source 11 and the installation position of the collimating lens 121 relatively fixed, ensuring that the collimating lens 121 can accurately process the divergent light into wide-band parallel light; secondly, the user can synchronously adjust the height of the optical fiber point light source 11 and the collimating lens 121 through the third adjustment component 125, and then change the imaging height of the microscopic multi-color image and the reflected light image to meet the acquisition requirements of the high-speed camera 5 and the color camera 3.

[0064] More specifically, if Figure 4 As shown, the third adjustment component 125 includes a base frame 1251, a mounting shaft 1252 mounted on the base frame 1251 with adjustable height, and a tightening bolt 1253 mounted on the base frame 1251 for tightening the mounting shaft 1252 to fix the height of the mounting shaft 1252, and the top of the mounting shaft 1252 is fixedly connected to the collimating lens frame 123.

[0065] In some preferred embodiments, the light source filter 13 is installed through the fourth adjustment component 126, and its installation height is adjustable, so that the light source filter 13 can match the fiber optic point light source 11 and the collimating lens 121 for height adjustment; wherein, the structure of the fourth adjustment component 126 is similar to the third adjustment component 125, so it is not repeated here.

[0066] In some preferred embodiments, a focusing lens assembly 7 is provided between the beam splitter prism 2 and the color camera 3 and between the dichroic mirror stack 4 and the high-speed camera 5 .

[0067] Specifically, the focusing lens assembly 7 has a focusing lens that can focus the corresponding input parallel light onto the target surface of the sensor of the high-speed camera 5 or the color camera 3. The former can focus and differentiate the narrow-band light beam to form a spectroscopic light spot, and the latter can form a colorful reflected light image.

[0068] In some preferred embodiments, the splitting ratio T:R of the beam splitting prism 2 is 5:5, that is, the incident light is divided into reflected light and transmitted light in a ratio of 50% to 50%, and its polarization state is not changed. This enables the reflected light image and the microscopic multi-color image to be imaged with light beams of the same light intensity, thereby improving the comparability of the two, and thereby improving the accuracy and reliability of the comparison results of the difference between the two.

[0069] In some preferred embodiments, a fifth adjustment component 8 for adjusting the relative distance between the beam splitter prism 2 and the light source filter 13 is provided at the bottom of the beam splitter prism 2, which can be used to adjust the emission direction of the reflected light, thereby changing the imaging position of the reflected light image.

[0070] Second, please refer to Figure 6 Some embodiments of the present application also provide a correction method for a color separation adjustment system of an image flow cytometer, which is applied in a color separation adjustment system of an image flow cytometer, and the system includes: A light source assembly 1, used to generate parallel light; A beam splitter prism 2, used for splitting parallel light into reflected light and transmitted light; A color camera 3, used for collecting reflected light to generate a reflected light image; A dichroic mirror stack 4, having a plurality of dichroic lenses 442 with adjustable orientation angles, for outputting a plurality of outgoing light beams of different wavelengths according to the transmitted light; A high-speed camera 5 is used to collect the outgoing light beam to generate multiple narrow-band outgoing light images; Processing module 6, used for processing the multi-segment narrow-band emission light images into microscopic multi-color images, so as to compare the difference with the reflected light images to realize color separation correction and adjustment; The method comprises the steps of: S1, adjusting the orientation angle of each dichroic lens 442 in the dichroic mirror stack 4 in turn based on the preset position and accuracy requirements, so as to correct and adjust the position of each narrow band pattern in the multi-segment narrow band output light image; S2, comparing the difference between the microscopic multi-color image after correction and adjustment and the corresponding reflected light image, if the difference is as expected, then end the correction, otherwise return to step S1.

[0071] Specifically, the multi-segment narrow-band emergent light image is a black-and-white image including a plurality of narrow-band graphics distinguished based on color separation. The processing module 6 can present the multi-segment narrow-band emergent light image in a manner that allows the user to know the specific imaging positions of the narrow-band graphics, and then determine whether the specific imaging positions of the narrow-band graphics are accurate and whether they meet the preset position and precision requirements; the processing module 6 can also present the multi-segment narrow-band emergent light image and a preset position indication frame in a manner that allows the user to intuitively know whether the specific imaging positions of the narrow-band graphics meet the preset position and precision requirements; the processing module 6 can also analyze the relationship between the specific imaging position of the narrow-band graphics and the preset position and precision requirements through a preset graphic analysis algorithm, and then automatically determine whether the specific imaging position of the corresponding narrow-band graphics meets the use requirements, and if not, output a corresponding correction adjustment indication according to the difference between the specific imaging position and the preset position, so as to inform the user of the adjustment direction and adjustment amount of the corresponding dichroic lens 442.

[0072] More specifically, step S1 adjusts the positions of the various color separation spots in a sequential manner, so that the narrow band patterns corresponding to the various color separation spots meet the imaging requirements.

[0073] More specifically, considering that each narrow-band graphic has a certain accuracy requirement, there are still certain errors between its actual specific imaging position and the preset position, and the microscopic multi-color color image obtained based on these narrow-band graphics processing will superimpose these errors, which may cause poor imaging quality. Therefore, the correction method of the image flow cytometer color separation adjustment system of the embodiment of the present application further introduces step S2 for image difference comparison, and analyzes the image difference based on the reflected light image that can represent the imaging effect of the actual color image as a unified correction standard, so as to perform a second verification of the color separation correction adjustment, and when the difference does not meet expectations, continue to return to step S1 for analysis, correction and adjustment again.

[0074] More specifically, the process of comparing the difference between the corrected and adjusted microscopic multi-color image and the corresponding reflected light image is preferably implemented by outputting the corresponding image similarity in real time according to the two types of images based on the aforementioned preset similarity algorithm or difference algorithm, and then determining whether the difference meets expectations by comparing the image similarity with the preset similarity threshold.

[0075] The correction method of the color separation adjustment system of the image flow cytometer of the embodiment of the present application realizes the color separation correction adjustment of the image flow cytometer color separation adjustment system by means of secondary verification. The method realizes the secondary verification by comparing the image difference between the reflected light image which is based on the imaging effect that can represent the actual color image and is used as a unified correction standard and the microscopic multi-color color image which is reconstructed based on the multi-segment narrow-band output light image after correction and adjustment, so as to intuitively evaluate the imaging quality of the microscopic multi-color color image. When it does not meet the expectations, the orientation angle of each dichroic lens 442 in the dichroic mirror stack 4 is adjusted based on this, so as to complete the color separation correction adjustment conveniently and intuitively, which can effectively improve the adjustment accuracy of the color separation correction of the flow array image acquisition system.

[0076] More specifically, in the process of step S2 returning to step S1, reference adjustment data can also be generated according to the image difference, so that the user can adjust the orientation of the corresponding dichroic lens 442 according to the reference adjustment data for correction adjustment. The reference adjustment data can be determined based on the position difference of different color separation data about the geometric center of the image, or it can be determined by taking a certain type of color separation data as a reference system and calculating the position difference of other color separations.

[0077] In some preferred embodiments, Figure 2 As shown, during the execution of step S1 , a micro-target 9 is disposed between the light source assembly 1 and the beam splitter prism 2 .

[0078] Specifically, the correction method of the image flow cytometer color separation adjustment system of the embodiment of the present application includes two correction methods, namely, setting a micro-target 9 and not setting a micro-target 9, and the difference lies in the difference in the correction objects of the reflected light image and the multi-segment narrow-band output light image. In the correction method without setting the micro-target 9, the correction object is the light spot formed by the output of the light source component 1, and in the correction method with setting the micro-target 9, the correction object is the micro-target 9; in the implementation method where the correction object is the light spot formed by the output of the light source component 1, the correction process generally uses the light spot contour as a reference figure (generally a circle) for correction and adjustment; in the implementation method where the correction object is the micro-target 9, the specific shape of the micro-target 9 is used as the reference figure.

[0079] More specifically, the light spot formed by the output of the light source assembly 1 may be of poor quality, which may easily lead to blurred or missing edges of the light spot, making it impossible to use it as a reference image. In these cases, the correction method of the color separation adjustment system of the image flow cytometer of the embodiment of the present application introduces the micro-target 9 as a reference image to ensure that the color separation correction adjustment process can proceed smoothly.

[0080] In some preferred embodiments, when step S2 is executed for a preset number of times, if the difference still does not meet expectations, the dichroic mirror stack 4 is replaced or the preset position is modified, and then the process returns to step S1.

[0081] Specifically, if multiple corrections still fail to pass the verification of step S2, it indicates that there is a problem with the configuration of the color separation adjustment system of the image flow cytometer in the current embodiment of the present application, which may be due to the mismatch of the specifications of the dichroic mirror stack 4 or the problem with the preset position positioning, so it is necessary to perform color separation correction adjustment after completing the corresponding adjustment.

[0082] In the description of this specification, the description with reference to the terms "one embodiment", "certain embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0083] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A color separation adjustment system for an image flow cytometer, characterized in that: The system comprises: A light source assembly, used for generating parallel light; A beam splitter prism, used to split the parallel light into reflected light and transmitted light; A color camera, used for collecting the reflected light to generate a reflected light image; A dichroic mirror stack, comprising a plurality of dichroic lenses with adjustable orientation angles, for outputting a plurality of outgoing light beams of different wavelengths according to the transmitted light; A high-speed camera, used for collecting the outgoing light beam to generate multiple narrow-band outgoing light images; The processing module is used to process the multiple narrow-band emission light images into microscopic multi-color images so as to compare the differences with the reflected light images to realize color separation correction and adjustment.

2. The color separation adjustment system of image flow cytometer according to claim 1, characterized in that: The dichroic mirror stack includes a base frame, an adjustment frame, a first adjustment assembly, and a plurality of adjustable lens assemblies; The adjustable lens assemblies are all mounted on the adjustment frame, the adjustment frame is mounted on the base frame, and the first adjustment assembly is used to adjust the position of the adjustment frame on the base frame to synchronously change the orientation angle of the adjustable lens assembly relative to the beam splitter prism; Each of the adjustable lens assemblies includes a dichroic lens with an adjustable orientation angle.

3. The color separation adjustment system of image flow cytometer according to claim 2, characterized in that: The adjustable lens assembly includes a lens clip, the dichroic lens and a second adjustment assembly, the dichroic lens is fixed on the lens clip, the lens clip is installed on the adjustment frame, and the second adjustment assembly is used to adjust the position of the lens clip on the adjustment frame to change the orientation angle of the corresponding dichroic lens relative to the beam splitter prism.

4. The color separation adjustment system of image flow cytometer according to claim 1, characterized in that: The process of processing the multiple narrow-band emission light images into microscopic multi-color images comprises: The multiple narrow-band emission light images are sequentially subjected to color separation, cropping, color restoration and merging processing to obtain the microscopic multi-color image.

5. The color separation adjustment system of image flow cytometer according to claim 1, characterized in that: The light source assembly comprises an optical fiber point light source, a light source collimating assembly and a light source filter which are arranged in sequence.

6. The color separation adjustment system of image flow cytometer according to claim 5, characterized in that: The light source collimation assembly comprises a collimating lens, an optical fiber fixing sleeve, a collimating lens frame, a fixing clamp ring and a third adjusting assembly; The optical fiber point light source is fixed on the optical fiber fixing sleeve, the optical fiber fixing sleeve is fixedly connected to the collimating lens frame, the collimating lens is fixed on the collimating lens frame through a fixing clamp, and the collimating lens frame is height-adjustably mounted on the third adjustment component.

7. The color separation adjustment system of image flow cytometer according to claim 1, characterized in that: A focusing lens assembly is provided between the beam splitter prism and the color camera and between the dichroic mirror stack and the high-speed camera.

8. A correction method for a color separation adjustment system of an image flow cytometer, applied in the color separation adjustment system of an image flow cytometer, characterized in that: The system comprises: A light source assembly, used for generating parallel light; A beam splitter prism, used to split the parallel light into reflected light and transmitted light; A color camera, used for collecting the reflected light to generate a reflected light image; A dichroic mirror stack, comprising a plurality of dichroic lenses with adjustable orientation angles, for outputting a plurality of outgoing light beams of different wavelengths according to the transmitted light; A high-speed camera, used for collecting the outgoing light beam to generate multiple narrow-band outgoing light images; A processing module, used for processing the multiple narrow-band emission light images into microscopic multi-color images, so as to compare the difference with the reflected light image to realize color separation correction and adjustment; The method comprises the steps of: S1. sequentially adjusting the orientation angles of the dichroic mirrors in the dichroic mirror stack based on the preset positions and accuracy requirements, so as to correct and adjust the positions of the narrow-band graphics in the multi-segment narrow-band emergent light images; S2, comparing the difference between the microscopic multi-color image after correction and adjustment and the corresponding reflected light image, if the difference is as expected, then end the correction, otherwise return to step S1.

9. The calibration method of the color separation adjustment system of the image flow cytometer according to claim 8, characterized in that: During the execution of step S1, a micro-target is provided between the light source assembly and the beam splitter prism.

10. The calibration method of the color separation adjustment system of the image flow cytometer according to claim 8, characterized in that: When step S2 is executed for a preset number of times, if the difference still does not meet expectations, the dichroic mirror stack is replaced or the preset position is modified, and then the process returns to step S1.

Images